KEYWORDS: Point spread functions, 3D metrology, Molecules, Engineering, Deep learning, Source mask optimization, Neural networks, Fluorescence, Signal to noise ratio, Polymers
Simultaneous measurement of the 3D orientation and the 3D position of a single fluorescent molecule can be achieved by Point Spread Function (PSF) engineering. However, this 5D problem is complex to optimize and time consuming when solved with classical approaches. To overcome this problem, we developed a deep learning approach that allows us to obtain an optimized phase mask as well as an Analysis Neural Network that estimates the 5 parameters of immobilized single molecules with a reduced computation time. Our method shows an axial precision of about 30 nm and an orientation precision of about 10 degrees, and it can be applied to complex problems such as molecular orientation in membranes.
Biological nano-objects rapidly diffuse in the solution phase, impeding our efforts to monitor their physical and chemical properties for extended periods of time. To overcome this, we developed the Interferometric Scattering Anti-Brownian ELectrokinetic (ISABEL) Trap, which counteracts Brownian motion for an extended time by tracking a particle’s location via its scattering and rapidly applying positional feedback with electrokinetic forces. Recently, we improved the flexibility of these experiments by shifting the scattering detection beam to the near-infrared and opening the visible region for flexible and specific fluorescence measurements. These capabilities allow us to monitor the physical and chemical properties of the carboxysome, a ~100nm bacterial microcompartment responsible for CO2 fixation by the enzyme Rubisco. With the ISABEL trap, we can rapidly interleave 405 and 488 nm fluorescence excitation beams to measure the redox properties inside individual carboxysomes using the redox reporting GFP mutant, roGFP2. The capabilities provided by the ISABEL trap allow us to design solution-phase single-particle experiments for a variety of biological nanoscale objects.
Anti-Brownian traps confine single molecules or particles in free solution by closed-loop feedback forces, allowing detailed characterization of photophysical and transport properties. We have recently extended this approach to tracking the particles with interferometric scattering at near infrared wavelengths. This extension allows trapping non-fluorescent particles and performing simultaneous modulated fluorescence measurements in the Interferometric Scattering Anti-Brownian ELectrokinetic (ISABEL) trap. Here we use the interferometric scattering signal in the ISABEL trap to measure the scattering cross-sections of single carboxysomes, bacterial nanocompartments involved in carbon fixation. With a core-shell model, we can calculate the total mass and internal loading of single nano-objects.
Abstract: Precision spectroscopic measurements of particles in free solution are greatly aided by use of an Anti-Brownian ELectrokinetic (ABEL) trap, which counteracts the effects of diffusion by means of closed-loop feedback to hold a single particle in a diffraction-limited spot for extended-time measurements. Generally, fluorescent emission from the trapped object is used to produce position estimates for the feedback circuit. However, many objects of interest may fluoresce only dimly (such as native fluorescence from a pigment-protein complex) or intermittently (such as a quantum dot). Here we report the development and demonstration of a new trapping modality that incorporates interferometric scattering to produce particle position estimates, called the Interferometric Scattering Anti-Brownian ELectrokinetic (ISABEL) trap. Using the ISABEL trap, we are able to completely decouple trapping from fluorescence detection, permitting trapping of dim and completely dark nanoparticles.
KEYWORDS: 3D image processing, Super resolution, Proteins, Interfaces, Stereoscopy, 3D metrology, Silicon, Microscopy, Luminescence, 3D image reconstruction
The organization and dynamics of the genome regulate DNA replication, DNA repair, and gene expression. Here, we developed a methodology for studies of 3D organization and dynamics of the nucleome over time scales ranging from milliseconds to hours, and length scales ranging from tens of nanometers to micrometers. We achieve unprecedented 3D track lengths throughout the mammalian nucleus by combining a novel labeling scheme using nanobody ArrayG/N fusions of dSpCas9 targeted to specific DNA loci; light sheet illumination for gentle imaging of live cells with high contrast; and point spread function engineering for parallel detection of multiple loci in 3D.
Anti-Brownian traps enable the measurement of single particles in free solution for long times by actively applying feedback forces based on an observed particle position to counteract Brownian motion. However, current implementations of anti-Brownian traps generally rely on fluorescence emission to detect a particle’s position. This reliance on fluorescence causes particles to be lost from the trap when they enter a fluorescence dark state by blinking or bleaching. Thus, there is a need for non-fluorescent methods of tracking for such traps. Scattered light provides a stable signal free of blinking and bleaching, but is very weak for small particles. However, interferometric scattering, a method of collecting the weak scattered field from a particle and interfering it with a strong reference field reflected from a nearby interface, allows particles to be tracked with sufficient speed and sensitivity. We combine interferometric scattering with our existing anti- Brownian electrokinetic (ABEL) trap to create the interferometric scattering anti-Brownian electrokinetic (ISABEL) trap. This technique enables the trapping of single nanoparticles in free solution for extended durations regardless of fluorescence blinking or bleaching. We verify the scaling of the interferometric scattering signal with the diameter of the particle for gold nanoparticles as small as 20 nm. We also demonstrate the measurement of the fluorescence brightness signal of fluorescent beads as they photobleach, while continuing to trap them with the scattering signal. The ISABEL trap extends the ability of anti-Brownian traps to new samples and new measurements across multiple scientific communities.
Single-molecule superresolution methods enable imaging of specifically-labeled biological samples with structures on length scales below the diffraction limit of visible light. Imaging samples at cryogenic temperatures (77 K) significantly reduces photobleaching, allowing more photons to be collected per emitter and thus improving the localization precision. Cryogenic single-molecule imaging also facilitates correlative imaging with cryogenic electron tomography (cryoET), which provides images of whole biological cells with high-resolution cellular contrast. Combining these two techniques by performing optical imaging under conditions that do not damage the sample for cryoET allows the combination of the high sensitivity and specificity from single-molecule fluorescence with the cellular context from cryoET. In this work, we use PAmKate, a red photoactivatable fluorescent protein, to perform cryogenic single-molecule imaging of proteins in the model organism Caulobacter crescentus at 77 K with sufficiently low illumination powers to prevent damage of the cryogenic sample. The enhanced number of photons detected allows localization precision to be improved to values below 10 nm.
To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an imaging platform that combines a novel, tilted light sheet illumination strategy with engineered long axial range point spread functions (PSFs) for low-background, 3D super localization of single molecules as well as 3D super-resolution imaging in thick cells. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The axial positions of the single molecules are encoded in the shape of the PSF rather than in the position or thickness of the light sheet, and the light sheet can therefore be formed using simple optics. The result is flexible and user-friendly 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D superresolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSF for fiducial bead tracking and live axial drift correction. We envision TILT3D to become an important tool not only for 3D super-resolution imaging, but also for live whole-cell single-particle and single-molecule tracking.
Point spread function (PSF) engineering has extended far-field localization microscopy into three dimensions by encoding the axial position of each emitter into the shape of its image on the detector. By fitting the observed PSF to a model function, one can extract position information with sub-diffraction precision. However, in practice this procedure is often complicated by optical aberrations present in the imaging system, which distort the shape of the observed PSF relative to the model function. The mismatch between the model and observed PSFs can limit the accuracy and precision achieved by the localization procedure.
Here, we present a simple method to experimentally improve the model PSF by phase retrieval of the pupil function of the imaging system using a set of images of an isolated emitter at different displacements from the focal plane. The pupil function is estimated by adding a phase term consisting of a combination of Zernike modes to the theoretical electric field at the back focal plane of the microscope. The amplitudes of the Zernike modes are determined by maximizing the likelihood function over all pixels in the experimental data set. Importantly, since all data is taken with the phase mask in place, we account for any aberrations it introduces. Using the resulting pupil function, we generate a model PSF which is significantly improved over the theoretical model in both the accuracy and precision of experimental emitter localizations. We also provide a MATLAB package which performs the entire fitting procedure, from phase retrieval to single-emitter localization.
We extend the information content of the microscope’s point-spread-function (PSF) by adding a new degree of freedom: spectral information. We demonstrate controllable encoding of a microscopic emitter’s spectral information (color) and 3D position in the shape of the microscope’s PSF. The design scheme works by exploiting the chromatic dispersion of an optical element placed in the optical path. By using numerical optimization we design a single physical pattern that yields different desired phase delay patterns for different wavelengths. To demonstrate the method’s applicability experimentally, we apply it to super-resolution imaging and to multiple particle tracking.
W. E. Moerner, the Harry S. Mosher Professor of Chemistry and professor, by courtesy, of Applied Physics at Stanford University, conducts research in physical chemistry of single molecules, biophysics, nanoparticle trapping, and nanophotonics. He earned three bachelor's degrees from Washington University in 1975 and master's and doctoral degrees from Cornell University in 1978 and 1982. From 1981 to 1995, he was a research staff member at IBM, receiving two IBM Outstanding Technical Achievement Awards. He was Professor and Distinguished Chair in Physical Chemistry at the University of California, San Diego, from 1995 to 1998, the year he joined the Stanford faculty.
Moerner received the 2014 Nobel Prize in Chemistry, along with Eric Betzig and Stefan Hell, for their development of super-resolved fluorescence microscopy.
Mean-squared displacement (MSD) analysis is one of the most prevalent tools employed in the application of single-particle tracking to biological systems. In camera-based tracking, the effects of “static error” due to photon fluctuations and “dynamic error” due to motion blur on the MSD have been well-characterized for the case of pure Brownian motion, producing a known constant offset to the straight-line MSD. However, particles tracked in cellular environments often do not undergo pure Brownian motion, but instead can for instance exhibit anomalous diffusion wherein the MSD curve obeys a power law with respect to time, MSD=2D*τα, where D* is an effective diffusion coefficient and 0 < α ≤ 1. There are a number of models that can explain anomalous diffusive behavior in different subcellular contexts. Of these models, fractional Brownian motion (FBM) has been shown to accurately describe the motion of labeled particles such as mRNA and chromosomal loci as they traverse the cytoplasm or nucleoplasm (i.e. crowded viscoelastic environments). Despite the importance of FBM in biological tracking, there has yet to be a complete treatment of the MSD in the presence of static and dynamic errors analogous to the special case of pure Brownian motion. We here present a closed-form, analytical expression of the FBM MSD in the presence of both types of error. We have previously demonstrated its value in live-cell data by applying it to the study of chromosomal locus motion in budding yeast cells. Here we focus on validations in simulated data.
Plants harvest sunlight by converting light energy to electron flow through the primary events in photosynthesis. One important question is how the light harvesting machinery adapts to fluctuating sunlight intensity. As a result of various regulatory processes, efficient light harvesting and photoprotection are balanced. Some of the biological steps in the photoprotective processes have been extensively studied and physiological regulatory factors have been identified. For example, the effect of lumen pH in changing carotenoid composition has been explored. However, the importance of photophysical dynamics in the initial light-harvesting steps and its relation to photoprotection remain poorly understood. Conformational and excited-state dynamics of multi-chromophore pigment-protein complexes are often difficult to study and limited information can be extracted from ensemble-averaged measurements. To address the problem, we use the Anti-Brownian ELectrokinetic (ABEL) trap to investigate the fluorescence from individual copies of light-harvesting complex II (LHCII), the primary antenna protein in higher plants, in a solution-phase environment. Perturbative surface immobilization or encapsulation schemes are avoided, and therefore the intrinsic dynamics and heterogeneity in the fluorescence of individual proteins are revealed. We perform simultaneous measurements of fluorescence intensity (brightness), excited-state lifetime, and emission spectrum of single trapped proteins. By analyzing the correlated changes between these observables, we identify forms of LHCII with different fluorescence intensities and excited-state lifetimes. The distinct forms may be associated with different energy dissipation mechanisms in the energy transfer chain. Changes of relative populations in response to pH and carotenoid composition are observed, which may extend our understanding of the molecular mechanisms of photoprotection.
F1-ATPase is the soluble portion of the membrane-embedded enzyme FoF1-ATP synthase that catalyzes the production of adenosine triphosphate in eukaryotic and eubacterial cells. In reverse, the F1 part can also hydrolyze ATP quickly at three catalytic binding sites. Therefore, catalysis of 'non-productive' ATP hydrolysis by F1 (or FoF1) must be minimized in the cell. In bacteria, the ε subunit is thought to control and block ATP hydrolysis by mechanically inserting its C-terminus into the rotary motor region of F1. We investigate this proposed mechanism by labeling F1 specifically with two fluorophores to monitor the C-terminus of the ε subunit by Förster resonance energy transfer. Single F1 molecules are trapped in solution by an Anti-Brownian electrokinetic trap which keeps the FRET-labeled F1 in place for extended observation times of several hundreds of milliseconds, limited by photobleaching. FRET changes in single F1 and FRET histograms for different biochemical conditions are compared to evaluate the proposed regulatory mechanism.
In aqueous solution, diffusion generally limits the observation window of a nano-meter sized single molecule to milliseconds and prevents quantitative determination of spectroscopic and transport properties molecule-by-molecule. The anti-Brownian electrokinetic (ABEL) trap is a feedback-based microfluidic device that enables prolonged (multiseconds) observation of single molecules in solution. The amount of information that can be extracted from each molecule in solution is thus boosted by three orders of magnitude. We describe recent advances in extending the ABEL trap to conduct both spectroscopic and transport measurements of single trapped molecules. First, by combining the trap with multi-parameter fluorescence detection, synchronized dynamics in different observables can be visualized in solution. We use single molecules of Atto 633 as an example and show that this popular label switches between different emissive states under common imaging conditions. Next, we show how transport properties of trapped single molecules can be extracted in addition to spectroscopic readouts. Due to their direct sensitivity to molecular size and charge, measured transport coefficients can be used to distinguish different molecular species and trace biomolecular interactions in solution. We demonstrate this new paradigm by monitoring DNA hybridization/melting in real-time.
We present a means of measuring the dipole orientation of a fluorescent, rotationally fixed single molecule (SM), using a specially designed phase mask, termed a “quadrated pupil,” conjugate to the back focal plane of a conventional widefield microscope. In comparison to image-fitting techniques that infer orientation by matching simulations to defocused or excessively magnified images, the quadrated pupil approach is more robust to minor modeling discrepancies, defocus, and optical aberrations. Precision on the order of 1°-5° is achieved in proofof- concept experiments for both azimuthal (φ) and polar (θ) angles. Since the phase mask is implemented on a liquid-crystal spatial light modulator (SLM) that may be deactivated without any mechanical perturbation of the sample or imaging system, the technique may be readily integrated into conventional imaging studies.
Single-molecule-based super-resolution fluorescence microscopy has recently been developed to surpass the diffraction
limit by roughly an order of magnitude. These methods depend on the ability to precisely and accurately measure the
position of a single-molecule emitter, typically by fitting its emission pattern to a symmetric estimator (e.g. centroid or
2D Gaussian). However, single-molecule emission patterns are not isotropic, and depend highly on the orientation of the
molecule’s transition dipole moment, as well as its z-position. Failure to account for this fact can result in localization
errors on the order of tens of nm for in-focus images, and ~50-200 nm for molecules at modest defocus. The latter range
becomes especially important for three-dimensional (3D) single-molecule super-resolution techniques, which typically
employ depths-of-field of up to ~2 μm. To address this issue we report the simultaneous measurement of precise and
accurate 3D single-molecule position and 3D dipole orientation using the Double-Helix Point Spread Function (DH-PSF)
microscope. We are thus able to significantly improve dipole-induced position errors, reducing standard deviations
in lateral localization from ~2x worse than photon-limited precision (48 nm vs. 25 nm) to within 5 nm of photon-limited
precision. Furthermore, by averaging many estimations of orientation we are able to improve from a lateral standard
deviation of 116 nm (~4x worse than the precision, 28 nm) to 34 nm (within 6 nm).
We use an Anti-Brownian ELectrokinetic (ABEL) trap to probe spectral emission shifts in solution-phase single
Peridinin-Chlorophyll-Proteins (PCPs). The ABEL trap allows localization of single biomolecules in solution in a small
volume for extended observation without immobilization. The essential idea combines fluorescence-based position
estimation with fast electrokinetic feedback in a microfluidic geometry to counter the Brownian motion of a single
nanoscale object, hence maintaining its position in a sub-micron-sized field of view for hundreds of milliseconds to
seconds. Peridinin-chlorophyll-protein is a water-soluble antenna protein found in dinoflagellates which
uses peridinins (carotenoids) as accessory light harvesting pigments to absorb sunlight in the green region of the
spectrum before transferring electronic excitation to chlorophyll. PCP is simpler than many other antenna complexes in
that there are only two chlorophyll pigments per monomer which do not form an exciton. We use the ABEL trap to study
single PCP monomers in solution for several seconds each. A significant fraction of the molecules show slow spectral
shifts (spectral diffusion) relative to the bulk PCP spectrum. This is the first spectral emission measurement conducted in
the ABEL trap.
The ABEL trap allows trapping of single biomolecules in solution for extended observation without immobilization. The
essential idea combines fluorescence-based position estimation with fast electrokinetic feedback in a microfluidic
geometry to counter the Brownian motion of a single nanoscale object, hence maintaining its position in the field of view
for hundreds of milliseconds to seconds. Such prolonged observation of single proteins allows access to slow dynamics,
as probed by any available photophysical observables. We have used the ABEL trap to study conformational dynamics
of the β2-adrenergic receptor, a key G-protein coupled receptor and drug target, in the absence and presence of agonist.
A single environment-sensitive dye reports on the receptor microenvironment, providing a real-time readout of
conformational change for each trapped receptor. The focus of this paper will be a quantitative comparison of the ligandfree
and agonist-bound receptor data from our ABEL trap experiments. We observe a small but clearly detectable shift in
conformational equilibria and a lengthening of fluctuation timescales upon binding of agonist. In order to quantify the
shift in state distributions and timescales, we apply nonparametric statistical tests to place error bounds on the resulting
single-molecule distributions.
By providing spatial localization on the nanometer scale, eliminating the need for ensemble averaging, and permitting
non-invasive intracellular investigations, single-molecule imaging has brought much insight to biophysics. A
particularly enticing application for single-molecule imaging is the capability to investigate live cells and to examine
structure and dynamics in the natural environment. To obtain true superresolution, control of the emission of the single
molecules provides a way to maintain a sparse concentration of emitters for any frame so that sequential imaging leads
to a final reconstruction with information beyond the optical diffraction limit. In this paper, we discuss several single-molecule-
based fluorescence methods that are possible, and indeed often enabled, by having live cell specimens.
Far-field super-resolution microscopy is a rapidly emerging method that is opening up opportunities for biological
imaging beyond the optical diffraction limit. We have implemented a Stimulated Emission Depletion (STED)
microscope to image single dye, cell, and tissue samples with 50-80 nm resolution. First, we compare the STED
performance imaging single molecules of several common dyes and report a novel STED dye. Then we apply STED to
image planar cell polarity protein complexes in intact fixed Drosophila tissue for the first time. Finally, we present a
preliminary study of the centrosomal protein Cep164 in mammalian cells. Our images suggest that Cep164 is arranged in
a nine-fold symmetric pattern around the centriole, consistent with findings suggested by cryoelectron tomography. Our
work demonstrates that STED microscopy can be used for superresolution imaging in intact tissue and provides
ultrastructural information in biological samples as an alternative to immuno-electron microscopy.
The point spread function (PSF) of a widefield fluorescence microscope is not suitable for three-dimensional superresolution
imaging. We characterize the localization precision of a unique method for 3D superresolution imaging
featuring a double-helix point spread function (DH-PSF). The DH-PSF is designed to have two lobes that rotate about
their midpoint in any transverse plane as a function of the axial position of the emitter. In effect, the PSF appears as a
double helix in three dimensions. By comparing the Cramer-Rao bound of the DH-PSF with the standard PSF as a
function of the axial position, we show that the DH-PSF has a higher and more uniform localization precision than the
standard PSF throughout a 2 μm depth of field. Comparisons between the DH-PSF and other methods for 3D superresolution
are briefly discussed. We also illustrate the applicability of the DH-PSF for imaging weak emitters in
biological systems by tracking the movement of quantum dots in glycerol and in live cells.
Recently, photoactivation and photoswitching were used to control single-molecule fluorescent labels and produce
images of cellular structures beyond the optical diffraction limit (e.g., PALM, FPALM, and STORM). While previous
live-cell studies relied on sophisticated photoactivatable fluorescent proteins, we show in the present work that
superresolution imaging can be performed with fusions to the commonly used fluorescent protein EYFP. Rather than
being photoactivated, however, EYFP can be reactivated with violet light after apparent photobleaching. In each cycle
after initial imaging, only a sparse subset fluorophores is reactivated and localized, and the final image is then generated
from the measured single-molecule positions. Because these methods are based on the imaging nanometer-sized single-molecule
emitters and on the use of an active control mechanism to produce sparse sub-ensembles, we suggest the
phrase "Single-Molecule Active-Control Microscopy" (SMACM) as an inclusive term for this general imaging strategy.
In this paper, we address limitations arising from physiologically imposed upper boundaries on the fluorophore
concentration by employing dark time-lapse periods to allow single-molecule motions to fill in filamentous structures,
increasing the effective labeling concentration while localizing each emitter at most once per resolution-limited spot.
We image cell-cycle-dependent superstructures of the bacterial actin protein MreB in live Caulobacter crescentus cells
with sub-40-nm resolution for the first time. Furthermore, we quantify the reactivation quantum yield of EYFP, and find
this to be 1.6 x 10-6, on par with conventional photoswitchable fluorescent proteins like Dronpa. These studies show that
EYFP is a useful emitter for in vivo superresolution imaging of intracellular structures in bacterial cells.
We have designed and studied the photophysics of a class of organic fluorophores termed "DCDHFs," which were
originally used as push-pull chromophores for nonlinear optical applications. In this paper, we describe the general
photophysics of many realizations of the DCDHF class of single-molecule emitters. Moreover, we have reengineered a
red-emitting DCDHF fluorophore so that it is dark until photoactivated with a short burst of low-intensity violet light.
Photoactivation of the dark fluorogen leads to conversion of an azide to an amine, which shifts the absorption to long
wavelengths. After photoactivation, the fluorophore is bright and photostable enough to be imaged on the singlemolecule
level in living cells. This molecule and its relatives will provide a new class of bright photoactivatable
fluorophores, as are needed for super-resolution imaging schemes that require active control of single-molecule
emission.
The hardware-based Anti-Brownian ELectrokinetic trap (ABEL trap) features a feedback latency as short as 25 μs,
suitable for trapping single protein molecules in aqueous solution. The performance of the feedback control loop is
analyzed to extract estimates of the position variance for various controller designs. Preliminary data are presented in
which the trap is applied to the problem of determining the distribution of numbers of ATP bound for single chaperonin
multi-subunit enzymes.
The Anti-Brownian Electrophoretic trap (ABEL trap) allows a user to trap and manipulate individual fluorescent molecules in solution. The heart of the ABEL trap is a microfluidic cell. In previous incarnations of the ABEL trap, the microfluidic cell was formed from a polydimethylsiloxane (PDMS) stamp and a glass coverslip. Here we present an improved microfluidic cell, made entirely out of glass. This new design significantly decreases the rate of photobleaching, which previously limited the time that a single molecule could be trapped. Chemical modifications to the surface of the cell prevent adsorption and allow one to control the balance between electroosmotic and electrophoretic forces. The depth of the trapping region in the cell can be adjusted to allow trapping of different-sized objects.
The Anti-Brownian ELectrophoretic trap (ABEL trap) is a new device that allows a user to trap and manipulate fluorescent objects as small as 20 nm freely diffusing in solution. We describe in detail how to build an ABEL trap.
Miniaturized and robust sensing modules are required for the development of portable integrated biological analysis systems or micro total analysis systems (μ-TAS). This work uses vertical cavity surface emitting lasers (VCSELs), optical emission filters and PIN photodetectors to realize a monolithically integrated, near-infrared, fluorescence detection system. The integration of these optoelectronic devices with biochips will drastically reduce cost of current systems and increase parallelism and portability. The sensor has been implemented on a micro-fluidic format, and sensitivity was evaluated. A theoretical limit of detection of IR-800 dye in methanol is reported to be 40 nM. The sensor sensitivity is limited by laser background as a result of integrating the optoelectronic elements in such close proximity. Significant reduction in laser background from reflections above the sensor is possible by increasing the distance between the sensor and optical interfaces to greater than 3 mm. Also, for distances greater than 500 microns between the sensor and optical interfaces above the sensor, it is found that background from indirect spontaneous emission is much smaller than the background caused by specular reflections of the laser.
A new class of fluorophores has been identified that can be imaged at the single-molecule level and offer additional beneficial properties such as a significant ground state dipole moment, moderate hyperpolarizability, and sensitivity to local rigidity. These molecules contain an amine donor and a dicyanodihydrofuran (DCDHF) acceptor linked by a conjugated unit (benzene, thiophene, alkene, styrene, etc.) and were originally designed to deliver both high polarizability anisotropy and dipole moment as nonlinear optical chromophores for photorefractive applications. Surprisingly, we have found that these molecules are also well-suited for single-molecule fluorescence imaging in polymers and other reasonably rigid environments. We report the bulk (ensemble) and single-molecule photophysical properties measured for six dyes in this new class of single-molecule reporters, with absorption maxima ranging from 486 to 614 nm.
We propose a new method to detect ultrasmall nonreciprocal phase shifts in solids based on the Sagnac interferometer combined with internal optical modulation of the absorption. The Sagnac interferometer with heterodyne balanced detection is expected to enable shot-noise-limited detection of phase shift as a result of its insensitivity to frequency and amplitude fluctuations of the laser. The low-concentration molecular sample is internally modulated by optical saturation with a pulsed laser. This internal modulation makes the molecular absorption time-dependent, and also removes the possibility of amplitude modulation feedthrough. We describe the design of this experiment and present preliminary characterizations of the noise performance.
Derivatives of 2-dicyanomethylen-3-cyano-2,5-dihydrofuran (DCDHF) have been synthesized by different methods to be used as photorefractive (PR) chromophores. Structure modifications were performed on the donor, acceptor and conjugated π-system for improving properties such as glass formation. Structure-property relationships important for PR applications are discussed from the results of studies including UV-Vis, electrochemistry and DSC.
Since the first observation of the photorefractive (PR) effect in polymers, extensive efforts have been directed toward understanding the physics of the PR process in these systems, as well as optimizing polymer composites and glasses for various applications. Despite remarkable progress both in elucidating the mechanisms and processes contributing to the PR effect and in designing organic materials with high gain and diffraction efficiency, simultaneously attaining high refractive index modulation, fast dynamics, and good thermal properties in one material remains a challenge. Monolithic glasses represent an attractive class of PR organic materials since they possess large nonlinearities and minimal inert volume, which enhances the performance without stability problems. In this paper, we present a complete study of monolithic glasses based on a promising new class of chromophores (containing 2-dicyanomethylen-3-cyano-5,5-dimethyl-2,5-dihydrofuran, abbreviated as DCDHF-derivatives). We describe thermal, photoconductive, orientational, and photorefractive properties of these materials in both red and near infrared wavelength regions. By studying the temperature dependence of various parameters, we analyze the factors that affect photorefractivity in DCDHF-based materials.
In this work we describe preliminary experiments in which we have used ultra-sensitive fluorescence microscopy to observe the dynamics of individual enzyme molecules acting upon a substrate. The enzyme, (beta) -galactosidase from E.coli, is specifically immobilized onto a glass substrate while maintaining its functionality. The immobilized protein degrades a fluorogenic substrate to produce a fluorescent product, whose generation can be observed in real time. Individual copies of (beta) -galactosidase can be observed for many minutes, allowing the measurement of a large number of successive substrate turnover events. A rudimentary analysis of these turnovers using autocorrelation functions is presented, and a strong heterogeneity in reaction rates between different molecules is observed. In addition, the challenges inherent in successful surface immobilization of proteins for single-molecule experiments are discussed.
We seek to improve the performance of organic photorefractive (OPR) systems by implementing two different design philosophies. In one strategy polysiloxane-based charge transport polymers are used to explore the requirement of a low glass transition temperature. These polymers allow construction of low Tg composites without plasticizcer and additionally may have higher charge mobility than poly(n-vinyl carbazole).The other strategy entails the use of small-molecule organic glasses composed of covalently attached charge transport and non-linrar optical chromophore moieties. Both classes of materials are characterized by holographic, photoconductive, and ellipsometric methods.
Photorefractive polymers have recently shown an attractive combination of high two-beam-coupling gain coefficient (approximately equals 200cm-1), low absorption (approximately equals 5-10 cm-1), and fast response (few ms) at 1W/cm2 writing intensity. Such materials show promise as adaptive beamsplitters for homodyne detection of transient phase shifts due to laser-based ultrasound. The performance of a photorefractive polymer composite is explored for this application.
In recent years, a number of adaptive interferometers have been used for the remote detection of laser-generated ultrasonic waves in parts under inspection. One type of adaptive interferometer uses two-wave mixing in a photorefractive material to form a real-time hologram, or adaptive beamsplitter. In this work we describe such an interferometric receiver based on two-wave mixing in photorefractive (PR) polymers. The PR polymers have a number of very promising technical features for this application which will be described in detail. In addition, the polymer composition can be tailored to specific requirements and the materials can be processed into a variety of different shapes and forms. Finally, when compared with inorganic photorefractive crystals, the polymers are inexpensive and simple to fabricate. We have taken advantage of the above features to demonstrate homodyne detection in the PR polymers with a sensitivity that is within a factor of three of the limit for an ideal homodyne system. We have also used a laboratory receiver to detect ultrasonic waves with good signal-to-noise. In this talk we will discuss the physical features of our polymer-based receiver and describe our experiments to characterize this system.
Daniel Wright, Anders Grunnet-Jepsen, Maria Diaz-Garcia, J. Casperson, B. Smith, Matthew Bratcher, Michael De Clue, Jay Siegel, William Moerner, Robert Twieg
A new and simple spectroscopic method for the determination of the trap density in C60-sensitized photorefractive polymers is presented. This method is compared with results obtained from traditional trap density measurements which involve two-wave mixing, and good correlation is observed. This technique is used to identify the inactive traps as the nonlinear optical chromophore. Also, composites with very low response times, high gain coefficients and low absorption are presented.
The development of fluorescence spectroscopy and imaging on the single molecule level has provided a new method to investigate various phenomena in condensed phase at liquid helium and room temperature unobscured by ensemble averaging. Single molecule spectroscopy allows studying the photophysical behavior of chromophores as well as using those chromophores as a probe for their local environment. A crucial problem that has to be overcome for single molecule studies in the liquid phase is Brownian motion: only partially or completely immobilized molecules allow extended observation. Here we report on the use of water-based polyacrylamide gels as a promising medium for single molecule investigations at room temperature with wide-field total internal reflection microscopy. The gel framework dramatically reduced Brownian motion of small fluorescent dye molecules. Observation of the diffusion of these molecules served as a probe for the inner structure of the gels. Furthermore these water-based gels form a useful medium for single molecule studies of biological systems in vitro.
This paper summarizes current research to develop photorefractive polymeric materials with improved speed, material stability, and high beam coupling gain. The recent demonstration of significantly improved two-beam coupling marks the entry into a gain regime which enables the observation of new effects for the first time, such as beam fanning and self-pumped phase-conjugation. These effects have previously been observed only in a few thick high gain inorganic photorefractive crystals. The large beam coupling forces the reinterpretation of such traditional characterization techniques such as the grating translation method for the determination of the spatial phase of the index grating. Our subsequent material study
focuses on several compositional variations to investigate the effect on the photorefractive performance of varying the chromophore and charge transporting polymer.
We describe on-going research to develop photorefractive polymeric materials with improved speed, material stability, and high beam coupling gain. The demonstration of significantly improved two-beam coupling is shown to mark the entry into a gain regime which enables the observation of new effects for the first time, such as beam fanning and self-pumped phase-conjugation. These effects have previously been reserved to a few thick high gain inorganic photorefractive crystals. We discuss how the large beam coupling has forced the reinterpretation of such traditional characterization techniques such as the grating translation method for the determination of the spatial phase of the index grating. Our subsequent material study focuses on several compositional variations to investigate the effect that varying the chromophore and charge transporting polymer has on the photorefractive effect.
We present a summary of the underlying mechanisms which govern the appearance and dynamics of the photorefractive effect in polymeric materials. Charge transport properties in polymers and their influence on the build-up of space- charge fields are discussed, and the beneficial effects from orientational enhancement of in situ room-temperature poling of nonlinear optical chromophores are reviewed. We introduce a new high performance low-glass-transition-temperature polymer composite and discuss its photorefractive properties as characterized by a thorough investigation of photoconductivity, two-wave beam coupling gain, grating phase shift, and diffraction efficiency as a function of intensity, applied electric field, and grating spacing.
Two experimental studies of new photorefractive polymer materials are described. First, an examination of the fabrication, stability, and photorefractive behavior of a recently-reported high-efficiency polymeric composite 33%PVK:50%DMNPAA:16%ECZ:1%TNF (K. Merrholz et at., Nature 371, 497) shows that this material is metastable, crystallizing on time scales from hours to days depending upon preparation. The grating phase shift also varies considerably from sample to sample. In the second part of this paper, first measurements and analysis of the properties of a new photorefractive polymer composite which contains a liquid nonlinear optical chromophore are presented. This new composite, which is immune to crystallization even at high chromophore loading levels, shows a useful diffraction efficiency of several percent at high fields, but the index of refraction grating is shown to consist of two components; a smaller photorefractive index grating and a grating which is not photorefractive in origin as shown by grating translation measurements.
Fluorescence excitation and vibrationally resolved dispersed fluorescence spectra of single molecules of terrylene in a polyethylene matrix at 1.5 K are presented. Single molecules are selected from the inhomogeneous ensemble by tuning a narrow-band dye laser into resonance with the sharp electronic origin features in highly dilute samples. Total fluorescence is detected with a photomultiplier and photon counting electronics, while vibrationally resolved fluorescence spectra are obtained by dispersing the emission onto a CCD detector. The excitation spectra reveal a variety of spectral diffusion effects on a wide range of time scales, including both continuous wandering over a range of frequencies and jumping among two or more discrete resonance frequencies. The dispersed fluorescence spectra show two distinct types of vibrational frequency and intensity patterns, possibly arising from terrylene molecules in the amorphous and crystalline regions of the polyethylene matrix, respectively. The description of the emission as resonance Raman or fluorescence is discussed.
A new class of photorefractive polymers is described in which an inert polymer binder is doped with a great molecule called a dual-function dopant, which has the dual functions of providing optical nonlinearity as well as charge transport as required for the photorefractive effect. These materials are the first photorefractive polymers to show both nondestructive readout as sufficiently low reading power and optical trap activation, in which pre-irradiation by a uniform light beam increases the concentration of deep trapping sites in the material. In the area of potential applications, a new sample configuration is described in which photorefractive polymers are fabricated into an electric field switchable stratified volume holographic structure. Individual layers may be activated by applied electric field leading to improved diffraction efficiency and angular selectivity.
Phase modulation and optical heterodyne detection were used to transmit and receive six cable television signals at 832 nm using a new polymeric electro-optic material. The electro-optic slab waveguide layer of the phase modulator consisted of a spin-coated, amorphous polyimide film doped with a nonlinear chromophore which had been poled in an electric field. The cladding layers, which confined the optical field within the waveguide, were composed of a photo-crosslinked acrylic polymer. Our television transmission system serves as a stringent test of the utility of the electro-optic polymer for lightwave data transmission.
Three nonlinear optical polymeric systems based on nonlinear optical chromophores attached as side chains to poly(methyl methacrylate) were synthesized and their nonlinear optical properties measured in thin film form by second harmonic generation and by the electro-optic effect. Also, attenuation of light propagation in optical waveguides and photothermal deflection infrared spectra were measured on these films to aid in selecting the best system for device development. Based on these results, a phase modulator was constructed and with sub- carrier multiplexing the signals from five TV channels were placed on an infrared laser beam as sidebands. After a short transmission through space, these signals were then heterodyne detected and displayed on a TV monitor with good signal to noise and with no crosstalk.
This paper describes the properties of a new class of materials exhibiting the photorefractive effect, doped nonlinear organic polymers. Photorefraction (at 647.1 nm) was established by a combination of hologram erasability, correlation with photoconductivity and electro-optic response, and enhancement by external fields in numerous samples (178 to 533 micrometers thick) of two nonlinear epoxy materials doped with hole transport agents based on p- diethylaminobenzaldehyde-diphenyl hydrazone (DEH). Diffraction efficiencies up to 0.1% were observed at bias fields near 100 kV/cm. A useful property of these materials is that poling of the nonlinear chromophores is partially reversible, permitting partial control of the grating readout independent of the space-charge field formed. The polarization anisotropy of grating readout is consistent with the photorefractive mechanism. Two-beam coupling measurements of both absorption and index gratings show (1) the absorption gratings are approximately 10 times smaller than the index gratings, and (2) the phase shift of the index grating is near 90 degree(s), which cannot occur via photochromism, heating, or any other process except photorefractivity.
Single-absorber optical spectroscopy in solids may be regarded as the problem of finding a single dopant impurity molecule in a 'haystack' composed of 1012 - 1018 background host molecules and up to $OM106 additional impurity molecules. Detailed studies of the low-temperature inhomogeneously broadened 0-0 S1 $IMP S0 electronic transition of pentacene dopant molecules in p-terphenyl crystals have yielded both (1) observations of spectral structure scaling as N, where N is the number of impurity molecules in resonance, and (2) the optical absorption spectrum of a single impurity molecule in a solid (N equals 1). Recent advances in fluorescence excitation of very small volumes have greatly improved the signal-to-noise ratio for a single molecule.
Jerome Swalen, Gary Bjorklund, Stephen Ducharme, William Fleming, Stephan Herminghaus, Dietmar Jungbauer, William Moerner, Barton Smith, Robert Twieg, Do Yoon, C. Grant Willson
Increased interest and activity in optoelectronics has resulted from anticipated applications
of optics to communication, electronic interconnections, information storage and possibly
even logic. Current nonlinear optical (NLO) devices today employ inorganic crystals, such
as lithium niobate for electrooptic modulation and switching and KDP or KNbO3 for
frequency doubling. Organic nonlinear optical materials, that is, poled organic polymeric
films and organic crystals, have the potential to replace some of these inorganic crystals.1
Key advantages include the high intrinsic nonlinearities of some organic molecules, the
ability to optimize the molecular structure for specific applications, low dc dielectric
constant, and low temperature processing. Current progress has also been made in
improving the long term stability of these materials. Here we wish to report on some of our
recent results on measurements of the order parameter, new crosslinked polymeric
materials which exhibit more stability and some device applications with polymeric
materials and organic crystals.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.